ROR1 and ROR2-novel targets for neuroblastoma.

Background: Despite advances in immunotherapeutic strategies for neuroblastoma (NBL), relapse remains a significant cause of mortality for high risk patients. The discovery of novel tumor associated antigens to improve efficacy and minimize the toxicities of immunotherapy is therefore warranted. Receptor Tyrosine Kinase-like Orphan Receptor-1 and 2 (ROR1 and ROR2) have been found to be expressed in several malignancies with limited expression in healthy tissues. Objectives: Given their role in tumor migration and proliferation and the fact that they were originally cloned from a NBL cell line, we hypothesized that ROR1 and ROR2 could serve as potential targets for anti-ROR1 and anti-ROR2 based immunotherapies in NBL. Methods: We characterized the mRNA and protein expression of ROR1 and ROR2 in NBL cell lines and tissue microarrays of patient samples. To explore the potential of ROR1 targeting, we performed in vitro cytotoxicity assays against NBL using NK92 cells as effector cells. Results: Both ROR1 and ROR2 are expressed across all stages of NBL. In patients with non-MYC amplified tumors, expression of ROR1/ROR2 correlated with survival and prognosis. Moreover, in a proof-of-concept experiment, pretreatment of NBL cell line with anti-ROR1 antibody showed additive cytotoxicity with NK92 cells. Conclusions: ROR1 and ROR2 could serve as novel targets for immunotherapy in NBL. The additive effect of anti-ROR1 antibodies with NK cells needs to be explored further to evaluate the possibility of combining anti-ROR1 antibodies with immune effectors such as NK92 cells as a potential off-the shelf immunotherapy for NBL and other ROR1 expressing malignancies.

Gammaherpesvirus infection and malignant disease in rhesus macaques experimentally infected with SIV or SHIV.

Human gammaherpesviruses are associated with malignancies in HIV infected individuals; in macaques used in non-human primate models of HIV infection, gammaherpesvirus infections also occur. Limited data on prevalence and tumorigenicity of macaque gammaherpesviruses, mostly cross-sectional analyses of small series, are available. We comprehensively examine all three-rhesus macaque gammaherpesviruses -Rhesus rhadinovirus (RRV), Rhesus Lymphocryptovirus (RLCV) and Retroperitoneal Fibromatosis Herpesvirus (RFHV) in macaques experimentally infected with Simian Immunodeficiency Virus or Simian Human Immunodeficiency Virus (SIV/SHIV) in studies spanning 15 years at the AIDS and Cancer Virus Program of the Frederick National Laboratory for Cancer Research. We evaluated 18 animals with malignancies (16 lymphomas, one fibrosarcoma and one carcinoma) and 32 controls. We developed real time quantitative PCR assays for each gammaherpesvirus DNA viral load (VL) in malignant and non-tumor tissues; we also characterized the tumors using immunohistochemistry and in situ hybridization. Furthermore, we retrospectively quantified gammaherpesvirus DNA VL and SIV/SHIV RNA VL in longitudinally-collected PBMCs and plasma, respectively. One or more gammaherpesviruses were detected in 17 tumors; generally, one was predominant, and the relevant DNA VL in the tumor was very high compared to surrounding tissues. RLCV was predominant in tumors resembling diffuse large B cell lymphomas; in a Burkitt-like lymphoma, RRV was predominant; and in the fibrosarcoma, RFHV was predominant. Median RRV and RLCV PBMC DNA VL were significantly higher in cases than controls; SIV/SHIV VL and RLCV VL were independently associated with cancer. Local regressions showed that longitudinal VL patterns in cases and controls, from SIV infection to necropsy, differed for each gammaherpesvirus: while RFHV VL increased only slightly in all animals, RLCV and RRV VL increased significantly and continued to increase steeply in cases; in controls, VL flattened. In conclusion, the data suggest that gammaherpesviruses may play a significant role in tumorogenesis in macaques infected with immunodeficiency viruses.

Cancer Incidence in C3H Mice Protected from Lethal Total-Body Radiation after Amifostine.

Amifostine is a potent antioxidant that protects against ionizing radiation effects. In this study, we evaluated the effect of Amifostine administered before total-body irradiation (TBI), at a drug dose that protects against TBI lethality, for potential protection against radiation-induced late effects such as a shortened lifespan and cancer. Three groups of mice were studied: 0 Gy control; 10.8 Gy TBI with Amifostine pretreatment; and 5.4 Gy TBI alone. Animals were monitored for their entire lifespan. The median survival times for mice receiving 0, 5.4 or 10.8 Gy TBI were 706, 460 and 491 days, respectively. Median survival of both irradiated groups was significantly shorter compared to nonirradiated mice ( P < 0.0001). Cancer incidence (hematopoietic and solid tumors) was similar between the irradiated groups and was significantly greater than for the 0 Gy controls. The ratio of hematopoietic-to-solid tumors differed among the groups, with the 5.4 Gy group having a higher incidence of hematopoietic neoplasms compared to the 10.8 Gy/Amifostine group (1.8-fold). Solid tumor incidence was greater in the 10.8 Gy/Amifostine group (1.6-fold). There are few mouse lifespan studies for agents that protect against radiation-induced lethality. Mice treated with 10.8 Gy/Amifostine yielded a lower incidence of hematopoietic neoplasms and higher incidence of solid neoplasms. In conclusion, mice protected from lethal TBI have a shortened lifespan, due in large part to cancer induction after exposure compared to nonexposed controls. Amifostine treatment did protect against radiation-induced hematopoietic tumors, while protection against solid neoplasms was significant but incomplete.

Interleukin-27 Is a Potential Rescue Therapy for Acute Severe Colitis Through Interleukin-10-Dependent, T-Cell-Independent Attenuation of Colonic Mucosal Innate Immune Responses.

BACKGROUND: If treatment with intravenous steroids fail, inflammatory bowel disease patients with acute severe colitis face systemic anti-tumor necrosis factor biologic rescue therapy or colectomy. Interleukin (IL)-27 is a cytokine with an immunosuppressive role in adaptive immune responses. However, the IL-27 receptor complex is also expressed on innate immune cells, and there is evidence that IL-27 can impact the function of innate cell subsets, although this particular functionality in vivo is not understood. Our aim was to define the efficacy of IL-27 in acute severe colitis and characterize novel IL-27-driven mechanisms of immunosuppression in the colonic mucosa. METHODS: We assessed oral delivery of Lactococcus lactis expressing an IL-27 hyperkine on the innate immune response in vivo in a genetically intact, noninfective, acute murine colitis model induced by intrarectal instillation of 2,4,6-trinitrobenzenesulfonic acid in SJL/J mice. RESULTS: IL-27 attenuates acute severe colitis through the reduction of colonic mucosal neutrophil infiltrate associated with a decreased CXC chemokine gradient. This suppression was T cell independent and IL-10 dependent, initially featuring enhanced mucosal IL-10. IL-27 was associated with a reduction in colonic proinflammatory cytokines and induced a multifocal, strong, positive nuclear expression of phosphorylated STAT-1 in mucosal epithelial cells. CONCLUSION: We have defined novel mechanisms of IL-27 immunosuppression toward colonic innate immune responses in vivo. Mucosal delivery of IL-27 has translational potential as a novel therapeutic for inflammatory bowel disease, and it is a future mucosal directed rescue therapy in acute severe inflammatory bowel disease.

Immunocompetent mouse allograft models for development of therapies to target breast cancer metastasis.

Effective drug development to combat metastatic disease in breast cancer would be aided by the availability of well-characterized preclinical animal models that (a) metastasize with high efficiency, (b) metastasize in a reasonable time-frame, (c) have an intact immune system, and (d) capture some of the heterogeneity of the human disease. To address these issues, we have assembled a panel of twelve mouse mammary cancer cell lines that can metastasize efficiently on implantation into syngeneic immunocompetent hosts. Genomic characterization shows that more than half of the 30 most commonly mutated genes in human breast cancer are represented within the panel. Transcriptomically, most of the models fall into the luminal A or B intrinsic molecular subtypes, despite the predominance of an aggressive, poorly-differentiated or spindled histopathology in all models. Patterns of immune cell infiltration, proliferation rates, apoptosis and angiogenesis differed significantly among models. Inherent within-model variability of the metastatic phenotype mandates large cohort sizes for intervention studies but may also capture some relevant non-genetic sources of variability. The varied molecular and phenotypic characteristics of this expanded panel of models should aid in model selection for development of antimetastatic therapies in vivo, and serve as a useful platform for predictive biomarker identification.

Adaptation of Laser Microdissection Technique for the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model by NanoString Technologies.

Laser capture microdissection (LCM) of tissue is an established tool in medical research for collection of distinguished cell populations under direct microscopic visualization for molecular analysis. LCM samples have been successfully analyzed in a number of genomic and proteomic downstream molecular applications. However, LCM sample collection and preparation procedure has to be adapted to each downstream analysis platform. In this present manuscript we describe in detail the adaptation of LCM methodology for the collection and preparation of fresh frozen samples for NanoString analysis based on a study of a model of mouse mammary gland carcinoma and its lung metastasis. Our adaptation of LCM sample preparation and workflow to the requirements of the NanoString platform allowed acquiring samples with high RNA quality. The NanoString analysis of such samples provided sensitive detection of genes of interest and their associated molecular pathways. NanoString is a reliable gene expression analysis platform that can be effectively coupled with LCM.

PET/CT imaging of renal cell carcinoma with (18)F-VM4-037: a phase II pilot study.

BACKGROUND: Carbonic anhydrase IX (CA-IX) is a potential imaging biomarker of clear cell renal cell carcinoma (ccRCC). Here, we report the results of a phase II clinical trial of a small molecule radiotracer targeting CA-IX ((18)F-VM4-037) in ccRCC. METHODS: Between October 2012 and May 2013, 11 patients with kidney masses underwent (18)F-VM4-037 PET/CT prior to surgery. Dynamic imaging was performed for the first 45 min post injection and whole-body imaging was obtained at 60 min post injection. Tumors were surgically excised or biopsied within 4 weeks of imaging. RESULTS: All patients tolerated the radiotracer well with no adverse events. Ten of the 11 patients had histologically confirmed malignancy. One patient had a Bosniak Type 3 cyst with no tumor found at surgery. Two patients had extrarenal disease and 9 had tumors only in the kidney. Primary ccRCC lesions were difficult to visualize on PET alone due to high uptake of the tracer in the adjacent normal kidney parenchyma, however when viewed in conjunction with CT, the tumors were easily localized. Metastatic lesions were clearly visible on PET. Mean SUV for primary kidney lesions was 2.55 in all patients; in patients with histologically confirmed ccRCC, the mean SUV was 3.16. The time-activity curves (TAC) are consistent with reversible ligand binding with peak activity concentration at 8 min post injection followed by washout. Distribution Volume Ratio (DVR) of the lesions was measured using the Logan graphical analysis method. The mean DVR value across the 9 kidney lesions was 5.2 ± 2.8, (range 0.68-10.34). CONCLUSION: 18F-VM4-037 is a well-tolerated PET agent that allows same day imaging of CA-IX expression. The agent demonstrated moderate signal uptake in primary tumors and excellent visualization of CA-IX positive metastases. While the evaluation of primary ccRCC lesions is challenging due to high background activity in the normal kidney parenchyma, 18F-VM4-037 may be most useful in the evaluation of metastatic ccRCC lesions.

Mass Spectrometry-Based Metabolomics Identifies Longitudinal Urinary Metabolite Profiles Predictive of Radiation-Induced Cancer.

Nonlethal exposure to ionizing radiation (IR) is a public concern due to its known carcinogenic effects. Although latency periods for IR-induced neoplasms are relatively long, the ability to detect cancer as early as possible is highly advantageous for effective therapeutic intervention. Therefore, we hypothesized that metabolites in the urine from mice exposed to total body radiation (TBI) would predict for the presence of cancer before a palpable mass was detected. In this study, we exposed mice to 0 or 5.4 Gy TBI, collected urine samples periodically over 1 year, and assayed urine metabolites by using mass spectrometry. Longitudinal data analysis within the first year post-TBI revealed that cancers, including hematopoietic, solid, and benign neoplasms, could be distinguished by unique urinary signatures as early as 3 months post-TBI. Furthermore, a distinction among different types of malignancies could be clearly delineated as early as 3 months post-TBI for hematopoietic neoplasms, 6 months for solid neoplasms, and by 1 year for benign neoplasms. Moreover, the feature profile for radiation-exposed mice 6 months post-TBI was found to be similar to nonirradiated control mice at 18 months, suggesting that TBI accelerates aging. These results demonstrate that urine feature profiles following TBI can identify cancers in mice prior to macroscopic detection, with important implications for the early diagnosis and treatment.

A melanin-independent interaction between Mc1r and Met signaling pathways is required for HGF-dependent melanoma.

Melanocortin 1 receptor (MC1R) signaling stimulates black eumelanin production through a cAMP-dependent pathway. MC1R polymorphisms can impair this process, resulting in a predominance of red phaeomelanin. The red hair, fair skin and UV sensitive phenotype is a well-described melanoma risk factor. MC1R polymorphisms also confer melanoma risk independent of pigment. We investigated the effect of Mc1r deficiency in a mouse model of UV-induced melanoma. C57BL/6-Mc1r+/+-HGF transgenic mice have a characteristic hyperpigmented black phenotype with extra-follicular dermal melanocytes located at the dermal/epidermal junction. UVB induces melanoma, independent of melanin pigmentation, but UVA-induced and spontaneous melanomas are dependent on black eumelanin. We crossed these mice with yellow C57BL/6-Mc1re/e animals which have a non-functional Mc1r and produce predominantly yellow phaeomelanin. Yellow C57BL/6-Mc1re/e-HGF mice produced no melanoma in response to UVR or spontaneously even though the HGF transgene and its receptor Met were expressed. Total melanin was less than in C57BL/6-Mc1r+/+-HGF mice, hyperpigmentation was not observed and there were few extra-follicular melanocytes. Thus, functional Mc1r was required for expression of the transgenic HGF phenotype. Heterozygous C57BL/6-Mc1re/+-HGF mice were black and hyperpigmented and, although extra-follicular melanocytes and skin melanin content were similar to C57BL/6-Mc1r+/+-HGF animals, they developed UV-induced and spontaneous melanomas with significantly less efficiency by all criteria. Thus, heterozygosity for Mc1r was sufficient to restore the transgenic HGF phenotype but insufficient to fully restore melanoma. We conclude that a previously unsuspected melanin-independent interaction between Mc1r and Met signaling pathways is required for HGF-dependent melanoma and postulate that this pathway is involved in human melanoma.

Telomere dysfunction suppresses multiple endocrine neoplasia in mice.

Multiple endocrine neoplasia (MEN) syndrome is typified by the occurrence of tumors in two or more hormonal tissues. Whereas the genetics of MEN syndrome is relatively well understood, the tumorigenic mechanisms for these cancers remain relatively obscure. The Cdk4 (R24C) mouse model develops highly penetrant pituitary tumors and endocrine pancreas adenomas, and, as such, this model is appropriate to gain insight into mechanisms underlying MEN. Using this model, here we provide evidence supporting an important role for telomerase in the pathogenesis of MEN. We observed increased aneuploidy in Cdk4 (R/R) fibroblasts along with significantly elevated telomerase activity and telomere length in Cdk4 (R/R) islets and embryonic fibroblasts. To better understand the role of telomerase, we generated Cdk4 (R24C) mice with inactivation of the mTERC locus, which codes for the essential RNA component of the enzyme telomerase (mTERC (-/-) Cdk4 (R/R) mice). Embryonic fibroblasts and islets derived from mTERC (-/-) Cdk4 (R/R) mice exhibit reduced telomere length and proliferative capacity. Further, mTERC (-/-) Cdk4 (R/R) fibroblasts display reduced transformation potential. Importantly, mTERC (-/-) Cdk4 (R/R) mice display significantly reduced spontaneous tumorigenesis. Strikingly, we observed dramatic suppression of pituitary tumors and endocrine pancreas adenomas in mTERC (-/-) Cdk4 (R/R) mice. Telomere dysfunction suppressed tumor initiation and increased latency of tumor development while not affecting the progression of established tumors. In summary, these results are suggestive of an important role for telomerase in tumor development in the Cdk4 (R24C) mouse model, specifically in the genesis of tumors in the pituitary and the endocrine pancreas.

Combination of 13-Cis retinoic acid and lovastatin: marked antitumor potential in vivo in a pheochromocytoma allograft model in female athymic nude mice.

Currently, there are no reliably effective therapeutic options for metastatic pheochromocytoma (PCC) and paraganglioma. Moreover, there are no therapies that may prevent the onset or progression of tumors in patients with succinate dehydrogenase type B mutations, which are associated with very aggressive tumors. Therefore, we tested the approved and well-tolerated drugs lovastatin and 13-cis-retinoic acid (13cRA) in vitro in an aggressive PCC mouse cell line, mouse tumor tissue-derived (MTT) cells, and in vivo in a PCC allograft nude mouse model, in therapeutically relevant doses. Treatment was started 24 hours before sc tumor cell injection and continued for 30 more days. Tumor sizes were measured from outside by caliper and sizes of viable tumor mass by bioluminescence imaging. Lovastatin showed antiproliferative effects in vitro and led to significantly smaller tumor sizes in vivo compared with vehicle treatment. 13cRA promoted tumor cell growth in vitro and led to significantly larger viable tumor mass and significantly faster increase of viable tumor mass in vivo over time compared with vehicle, lovastatin, and combination treatment. However, when combined with lovastatin, 13cRA enhanced the antiproliferative effect of lovastatin in vivo. The combination-treated mice showed slowest tumor growth of all groups with significantly slower tumor growth compared with the vehicle-treated mice and significantly smaller tumor sizes. Moreover, the combination-treated group displayed the smallest size of viable tumor mass and the slowest increase in viable tumor mass over time of all groups, with a significant difference compared with the vehicle- and 13cRA-treated group. The combination-treated tumors showed highest extent of necrosis, lowest median microvessel density and highest expression of α-smooth muscle actin. The combination of high microvessel density and low α-smooth muscle actin is a predictor of poor prognosis in other tumor entities. Therefore, this drug combination may be a well-tolerated novel therapeutic or preventive option for malignant PCC.

IFN-gamma AU-rich element removal promotes chronic IFN-gamma expression and autoimmunity in mice.

We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3' untranslated region (3'UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) (-/-) present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del(-/-) mice display increased numbers of pDCs in bone marrow and spleen. Addition of IFN-γ to Flt3-ligand (Flt3L) treated in vitro bone marrow cultures results in a 2-fold increase in pDCs with concurrent increases in IRF8 expression. Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-Del(-/-) mice. ARE-Del(+/-) mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del(+/-) mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-γ milieu on B220(+) cell types and in particular the impact of MZB cell loss on MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del(-/-) mice and SLE patients suggest that IFN-γ may not only be a product of SLE but may be critical for disease onset and progression.

Sporadic naturally occurring melanoma in dogs as a preclinical model for human melanoma.

Melanoma represents a significant malignancy in humans and dogs. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant preclinical model. Canine melanomas rarely arise in sun-exposed sites. Most occur in the oral cavity, with a subset having intra-epithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma. The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ-line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs. Canine and human mucosal melanomas appear to harbor BRAF, NRAS, and c-kit mutations uncommonly, compared with human cutaneous melanomas, although both species share AKT and MAPK signaling activation. We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas. This represents opportunity to explore canine oral cavity melanoma as a preclinical model.

Oral delivery of IL-27 recombinant bacteria attenuates immune colitis in mice.

BACKGROUND & AIMS: Treatment of inflammatory bowel disease would benefit from specific targeting of therapeutics to the intestine. We developed a strategy for localized delivery of the immunosuppressive cytokine interleukin (IL)-27, which is synthesized actively in situ by the food-grade bacterium Lactococcus lactis (LL-IL-27), and tested its ability to reduce colitis in mice. METHODS: The 2 genes encoding mouse IL-27 were synthesized with optimal codon use for L lactis and joined with a linker; a signal sequence was added to allow for product secretion. The construct was introduced into L lactis. Colitis was induced via transfer of CD4(+)CD45RB(hi) T cells into Rag(-/-) mice to induce colitis; 7.5 weeks later, LL-IL-27 was administered to mice via gavage. Intestinal tissues were collected and analyzed. RESULTS: LL-IL-27 administration protected mice from T-cell transfer-induced enterocolitis and death. LL-IL-27 reduced disease activity scores, pathology features of large and small bowel, and levels of inflammatory cytokines in colonic tissue. LL-IL-27 also reduced the numbers of CD4(+) and IL-17(+) T cells in gut-associated lymphoid tissue. The effects of LL-IL-27 required production of IL-10 by the transferred T cells. LL-IL-27 was more effective than either LL-IL-10 or systemic administration of recombinant IL-27 in reducing colitis in mice. LL-IL-27 also reduced colitis in mice after administration of dextran sodium sulfate. CONCLUSIONS: LL-IL-27 reduces colitis in mice by increasing the production of IL-10. Mucosal delivery of LL-IL-27 could be a more effective and safer therapy for inflammatory bowel disease.

Insulin-dependent diabetes induced by pancreatic beta cell expression of IL-15 and IL-15Rα.

Increased serum levels of IL-15 are reported in type 1 diabetes (T1D). Here we report elevated serum soluble IL-15Rα levels in human T1D. To investigate the role of IL-15/IL-15Rα in the pathogenesis of T1D, we generated double transgenic mice with pancreatic ß-cell expression of IL-15 and IL-15Rα. The mice developed hyperglycemia, marked mononuclear cell infiltration, ß-cell destruction, and anti-insulin autoantibodies that mimic early human T1D. The diabetes in this model was reversed by inhibiting IL-15 signaling with anti-IL2/IL15Rß (anti-CD122), which blocks IL-15 transpresentation. Furthermore, the diabetes could be reversed by administration of the Janus kinase 2/3 inhibitor tofacitinib, which blocks IL-15 signaling. In an alternative diabetes model, nonobese diabetic mice, IL15/IL-15Rα expression was increased in islet cells in the prediabetic stage, and inhibition of IL-15 signaling with anti-CD122 at the prediabetic stage delayed diabetes development. In support of the view that these observations reflect the conditions in humans, we demonstrated pancreatic islet expression of both IL-15 and IL-15Rα in human T1D. Taken together our data suggest that disordered IL-15 and IL-15Rα may be involved in T1D pathogenesis and the IL-15/IL15Rα system and its signaling pathway may be rational therapeutic targets for early T1D.

Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody.

Reactive oxygen species generated by NADPH oxidase 5 (Nox5) have been implicated in physiological and pathophysiological signaling pathways, including cancer development and progression. However, because immunological tools are lacking, knowledge of the role of Nox5 in tumor biology has been limited; the expression of Nox5 protein across tumors and normal tissues is essentially unknown. Here, we report the characterization and use of a mouse monoclonal antibody against a recombinant Nox5 protein (bp 600-746) for expression profiling of Nox5 in human tumors by tissue microarray analysis. Using our novel antibody, we also report the detection of endogenous Nox5 protein in human UACC-257 melanoma cells. Immunofluorescence, confocal microscopy, and immunohistochemical techniques were employed to demonstrate Nox5 localization throughout UACC-257 cells, with perinuclear enhancement. Tissue microarray analysis revealed, for the first time, substantial Nox5 overexpression in several human cancers, including those of prostate, breast, colon, lung, brain, and ovary, as well as in malignant melanoma and non-Hodgkin lymphoma; expression in most nonmalignant tissues was negative to weak. This validated mouse monoclonal antibody will promote further exploration of the functional significance of Nox5 in human pathophysiology, including tumor cell growth and proliferation.

Selenium and selenoprotein deficiencies induce widespread pyogranuloma formation in mice, while high levels of dietary selenium decrease liver tumor size driven by TGFα.

Changes in dietary selenium and selenoprotein status may influence both anti- and pro-cancer pathways, making the outcome of interventions different from one study to another. To characterize such outcomes in a defined setting, we undertook a controlled hepatocarcinogenesis study involving varying levels of dietary selenium and altered selenoprotein status using mice carrying a mutant (A37G) selenocysteine tRNA transgene (Trsp(tG37) ) and/or a cancer driver TGFα transgene. The use of Trsp(tG37) altered selenoprotein expression in a selenoprotein and tissue specific manner and, at sufficient dietary selenium levels, separate the effect of diet and selenoprotein status. Mice were maintained on diets deficient in selenium (0.02 ppm selenium) or supplemented with 0.1, 0.4 or 2.25 ppm selenium or 30 ppm triphenylselenonium chloride (TPSC), a non-metabolized selenium compound. Trsp(tG37) transgenic and TGFα/Trsp(tG37) bi-transgenic mice subjected to selenium-deficient or TPSC diets developed a neurological phenotype associated with early morbidity and mortality prior to hepatocarcinoma development. Pathology analyses revealed widespread disseminated pyogranulomatous inflammation. Pyogranulomas occurred in liver, lungs, heart, spleen, small and large intestine, and mesenteric lymph nodes in these transgenic and bi-transgenic mice. The incidence of liver tumors was significantly increased in mice carrying the TGFα transgene, while dietary selenium and selenoprotein status did not affect tumor number and multiplicity. However, adenoma and carcinoma size and area were smaller in TGFα transgenic mice that were fed 0.4 and 2.25 versus 0.1 ppm of selenium. Thus, selenium and selenoprotein deficiencies led to widespread pyogranuloma formation, while high selenium levels inhibited the size of TGFα-induced liver tumors.

Interleukin-6 is essential for primary resistance to Francisella tularensis live vaccine strain infection.

We employed Francisella tularensis live vaccine strain (LVS) to study mechanisms of protective immunity against intracellular pathogens and, specifically, to understand protective correlates. One potential molecular correlate identified previously was interleukin-6 (IL-6), a cytokine with pleotropic roles in immunity, including influences on T and B cell functions. Given its role as an immune modulator and the correlation with successful anti-LVS vaccination, we examined the role IL-6 plays in the host response to LVS. IL-6-deficient (IL-6 knockout [KO]) mice infected with LVS intradermally or intranasally or anti-IL-6-treated mice, showed greatly reduced 50% lethal doses compared to wild-type (WT) mice. Increased susceptibility was not due to altered splenic immune cell populations during infection or decreased serum antibody production, as IL-6 KO mice had similar compositions of each compared to WT mice. Although LVS-infected IL-6 KO mice produced much less serum amyloid A and haptoglobin (two acute-phase proteins) than WT mice, there were no other obvious pathophysiological differences between LVS-infected WT and IL-6 KO mice. IL-6 KO or WT mice that survived primary LVS infection also survived a high-dose LVS secondary challenge. Using an in vitro overlay assay that measured T cell activation, cytokine production, and abilities of primed splenocytes to control intracellular LVS growth, we found that IL-6 KO total splenocytes or purified T cells were slightly defective in controlling intracellular LVS growth but were equivalent in cytokine production. Taken together, IL-6 is an integral part of a successful immune response to primary LVS infection, but its exact role in precipitating adaptive immunity remains elusive.

IKK-ε coordinates invasion and metastasis of ovarian cancer.

Inhibitor of IκB kinases (IKK) are key regulators of NF-κB signaling. Three IKK isoforms-α, ß, and ε-have been linked to oncogenesis, yet the precise components of NF-κB signaling in ovarian cancer have not yet been dissected. We surveyed 120 ovarian cancer specimens for IKK-ε expression. Notably, cytoplasmic expression was elevated in metastatic lesions relative to primary tumors (P = 0.03). Therefore, we hypothesized that IKK-ε drives ovarian cancer metastasis. IKK-ε was identified previously as a breast cancer oncogene and was associated with poor clinical outcome in ovarian cancer. We now define an ovarian cancer-specific IKK-ε-regulated gene expression signature using stably expressed short hairpin RNA targeting IKK-ε. Pathway analysis of the signature indicated that IKK-ε regulates expression of genes involved in cell motility and inflammation. We further showed that IKK-ε depletion in metastatic ovarian cancer cell lines decreased growth, adhesion, and invasion. Consistently, human xenografts depleted of IKK-ε in mice showed decreased aggressiveness, whereas overexpression of IKK-ε in a less invasive ovarian cancer cell line increased metastasis in vivo. Taken together, these data provide evidence that IKK-ε is a key coordinator of invasion and metastasis programs in ovarian cancer. Inhibition of IKK-ε signaling thus emerges as a viable therapeutic strategy in women whose ovarian cancer shows aberrant activation of this pathway.

The antioxidant tempol reduces carcinogenesis and enhances survival in mice when administered after nonlethal total body radiation.

There is significant interest in the development of agents that can ameliorate radiation damage after exposure to radiation has occurred. Here we report that chronic supplementation of the antioxidant Tempol in the diet of mice can reduce body weight without toxicity, decrease cancer, and extend survival when administered after nonlethal total body radiation (TBI). These effects were apparent in two different strains of mice (C3H, CBA) exposed to TBI (3 Gy). Notably, delaying administration of the Tempol diet one month after TBI could also enhance survival. Tempol reduced the incidence of hematopoietic neoplasms (lymphomas) in both strains, whereas both the onset and incidence of nonhematopoietic neoplasms were reduced in CBA mice. These results encourage further study of Tempol as a chemopreventive, to reduce the incidence of radiation-induced second malignancies after a course of definitive radiation therapy. Tempol may also find applications to reduce the risk of cancers in populations exposed to nonlethal radiation due to nuclear accidents or terrorist attacks.